Field of the Disclosure
[0001] The present disclosure is directed to the culturing of biological cells. More particularly,
the present disclosure is directed to automated systems and methods for the culturing
of biological cells. Even more particularly, the present disclosure is directed to
methods and systems for monitoring and controlling the conditions of a biological
cell culture in a stand-alone culturing system.
Background
[0002] The culturing of cells is common in the field of biotechnology. Cell culturing refers
to the growing of biological cells in a controlled environment. In cell culturing,
cells of interest that have been isolated from living tissue are placed in a controlled
environment for a period of time where they are maintained and grown for later use
in, for example, a therapy. The cells are placed in a vessel such as a flask or a
Petri dish and maintained in a suitable environment which typically includes a liquid,
gel or other medium that provides the cells with necessary nutrients for cell growth.
[0003] In addition to providing the cells with a nutrient-containing medium, the cells must
also reside in a suitable gas and temperature environment. Gases such as CO
2 and O
2 must be carefully supplied (or removed) to allow the cells to grow. Furthermore,
factors such as pH, media volume must also be monitored and regulated to ensure suitable
cell growth.
[0004] Traditionally, the culturing of cells has been carried out in a suitable vessel,
such as the previously mentioned flask or dish, and kept in a temperature, humidity
and CO
2 controlled incubator for a required period of time. As the cells grow, they tend
to "outgrow" these traditional and small vessels and must often be "split" when the
cells achieve a pre-determined cell concentration of culture saturation. "Splitting"
of cells often requires that medium be moved and added and will, in general, increase
handling frequency, labor and the possible risk of contamination and/or cell loss.
[0005] More recently, cell culturing vessels such as those described in
U.S. Patent 9,255,243 have been developed which reduce the need to split cells. Vessels such as those described
in
U.S. Patent 9,255,243allow for a sufficient volume of culture medium to be added to the vessel over the
course of the culturing process, thereby avoiding the need for cell splitting and
some of the accompanying manual and handling systems that may otherwise be required.
Vessels of the type shown and described in
U.S. Patent 9,255,243 include a gas-permeable membrane at the bottom end of the vessel through which gases
such as CO
2 and O
2 enter and exit the vessel chamber.
[0006] While vessels such those described in
U.S. Patent 9,255,243 have simplified the culturing process to a degree, the culturing must still take
place in a traditional humidity-, temperature-, and CO
2-controlled incubator. Moreover, most operations associated with the culturing of
cells such as loading the vessel with cells, media, culture additives (cytokines,
supplements, antibiotics, etc.), culture sampling, cell monitoring and cell harvesting
must be performed manually. For example, the addition of culture reagents to the culture
vessel may be triggered only following the drawing and analysis of a manually collected
sample.
[0007] Thus, it would be desirable to provide a system and method of cell culturing that
does not need to be performed in its entirety or at all, in a traditional humidity-,
temperature-, and CO2-controlled incubator. In other words, it would be desirable
to provide a "stand-alone" cell culturing system. The term "stand-alone" generally
refers to a system or method that is independent of traditional incubation wherein
the vessel need not reside, and the cell culturing need not be carried out in a traditional
incubator.
[0008] It would also be desirable to provide a more automated system and method of biological
cell culturing wherein certain operations such as the delivery or loading of the vessel
with cells, media, culturing additives need not be manually performed but can instead
be pre-programmed into the system's control system to be carried out at a preselected
time. It would also be desirable to provide systems and methods wherein the conditions
within the culture vessel can be monitored and controlled to provide a suitable culturing
environment for a given cell population. Based on such monitoring, conditions may
be automatically adjusted or corrected, as needed. For example, it would be desirable
to monitor the cell concentration/number, temperature, pressure, and/or gas content
in the vessel and adjust such conditions accordingly.
[0009] The systems and methods described herein address these needs.
Summary
[0010] There several aspects to the subject matter described herein.
[0011] In one aspect, a system for the culturing of biological cells is described. The system
includes a culturing vessel having a top cap and a bottom support member defining
a culture chamber wherein the bottom support member includes a gas permeable membrane.
The system further includes a base assembly configured to receive the bottom support
member in a gas-tight, mating manner. The base assembly further includes one or more
conduits for delivering to and/or removing one or more gases from the base. The system
includes a culture medium source and a control system that has one or more sensors
for sensing at least one of temperature or pressure during cell culturing.
[0012] In another aspect, an automated method for culturing biological cells is disclosed.
The method includes monitoring certain conditions within a culturing vessel having
a gas-permeable membrane at the base of said vessel and automatically adjusting the
conditions based on said monitoring.
[0013] In a more particular aspect, a method for automatically controlling the conditions
of a biological cell culturing environment is disclosed. The method includes placing
one or more sensors at the interface of a culturing vessel and base docking unit for
receiving the vessel and communicating selected conditions detected by said sensors
to a controller coupled to said base docking unit. The method includes adjusting,
as necessary, one or more conditions of said culturing environment based on the detected
selected conditions.
Brief Description of the Drawings
[0014]
Figure 1 is a schematic view of a cell culture system as described herein;
Figure 2 is a schematic view of the cell culture vessel of Fig.1 with the lower collar
shown in cross-section; and
Figure 3 is a diagram of a control system for the system described herein.
Detailed Description of the Embodiments
[0015] The embodiments disclosed herein are for the purpose of providing a description of
the present subject matter, and it is understood that the subject matter may be embodied
in various other forms and combinations not shown in detail.
[0016] Figure 1 is a schematic view of system 10 described herein. As discussed above, system
10 may be a "stand-alone" system that does not require a traditional incubator for
maintaining and controlling the culturing of the biological cells. Alternatively,
the system may allow for certain steps in the overall cell culturing process to be
carried out in a traditional incubator while allowing other steps to be performed
outside of the incubator under the direction of the integrated controller of system
10. Thus, for example, system 10, as described in greater detail below, may be automated
and include an integrated controller for monitoring the culturing conditions and delivering
and removing fluid at predetermined times and/or in response to certain conditions.
[0017] As shown in Figure 1, system 10 includes a culture vessel 12. Culture vessel 12 includes
a lower collar 14 and a top cap 16. Collar 14 includes or carries a gas-permeable
membrane 18 and a mesh support 21, as shown in Figure 2. Vessel 12 is preferably cylindrical
and includes wall 17, preferably transparent, which together with collar 14 (and membrane
18) and cap 16 define the chamber 19 of vessel 12. Vessel 12 preferably is made of
a plastic or polymeric material and is preferably disposable. Examples of specific
dimensions, volumes and materials of vessel 12 may be found in, for example, in
U.S. Patent No. 9,255,243.
[0018] Collar 14 of vessel 12 may be configured to allow for direct attachment to base docking
unit 20 shown in Figure 1. Attachment of collar 14 (and thus vessel 12) may be by
any means that provide in air-tight seal between collar 14 and base docking unit 20.
By air-tight, it is meant that gas cannot escape from base docking unit 20 to the
outside environment. (Of course, the transfer of gases between base docking unit 20
and chamber 19 of vessel 12 through membrane 18 is intentional and desired.) In one
embodiment, mating of vessel 12 to base docking unit 20 may be through a locking arrangement
as generally shown in Figure 2. For example, vessel 12 may include a rigid support
member 60 at the bottommost end of vessel 12. Rigid support member 60 includes a circumferential,
downwardly extending tongue that mates with a circumferential sealing member 64 in
base docking unit 20. A clip or other retainer 66 may be used to secure vessel 12
to base docking unit 20. As shown in Fig. 2, clip 66 catches shoulder 68 in collar
14 while the other end of clip 66 is held by base docking unit 20. Other means for
securing vessel 12 to base docking unit 20 in an air-tight manner may also be used.
[0019] Base docking unit 20 may be portable and suitable for direct placement on a flat
surface. As shown in Figure 1, base docking unit 20 may be attached to and mechanically
coupled to agitation assembly 22. In one embodiment, as shown in Fig. 1, base docking
unit 20 may be mounted to a cam (or series of cams) and motor(s) in docking unit 20.
The motor(s), once activated would cause docking base unit 20 to move in a linear
or circular motion, thereby gently mixing the contents of vessel 12. In addition,
system 10 may also include a pivot 72 that allows tilting (manual or automated) of
base docking unit 20 and, more specifically, vessel 12 to aid in the harvesting of
cells. Tilting of vessel 12 directs cells to flow toward a corner of the vessel where
the "deepest" tube 30c has its terminal open end. This allows for more complete drawing
of fluid out of vessel 12 during the harvesting of cells. Accordingly, system 10 may
include a pivotable plate 70 to which base docking unit 20 is attached. Plate 70 may
be tilted (typically less than 45° and preferably up to about 30°) about pivot 72.
[0020] As further shown in Figure 1, system 10 will include media source 24 and may further
include additional containers 25 of fluids used during the culturing process such
as the cells themselves or culture additives. System 10 may further include one or
more pumps 26 for delivering the culture media, cells or other fluids to vessel 12.
Fig. 1 shows several variants of "pumps" that may be used, either separately or in
combination in the system disclosed herein. For example, pumps 26 may be syringes
which draw fluid from media source 24 or, alternatively may be prefilled as shown
by syringe 26' with the appropriate media source or other liquid. In another alternative
example, pumps 26" may be traditional peristaltic pumps or other pumps including compressible
diaphragms or plates. Whether the pumping units are syringes, peristaltic pumps or
other types of pumps, they may be mounted on a device panel 76 or console as shown
schematically in Figure 1.
[0021] As noted above, while Fig. 1 shows syringes 26 or 26', mounted on panel 76, it will
be understood that other types of pumps described herein such as peristaltic pumps
26" may likewise be mounted on console/panel 76. In addition, containers 24 and 25
may be suspended from hooks or hangers on console 76 or from a separate IV pole. Where
containers 24 and 25 are suspended from hooks/hangers on console 76, such hooks may
be coupled to weight scales that can monitor changes in weight in the containers.
As further shown in Figure 1, pumps and media sources 24 and containers 25 of other
liquids communicate with the chamber 19 of vessel 12 by flexible tubing 30 which define
flow paths from the containers or pumps (syringes) and extend through channels 32
in cap 16 of vessel 12. Tubings 30a-30c define flow paths for delivering and withdrawing
liquids to and from the chamber of vessel 12. Tubings 30 may be of varying length,
with tubing 30c extending most deeply into chamber and is used for harvesting cells
with or without tilting of vessel 12, as described above. In an alternative, vessel
12 may include tubing segments that extend out of cap 16 and into chamber 19 of vessel
12. These tubing segments may be joined, in a known sterile fashion, to tubing segments
associated with the containers/syringes of media and other liquids. The sterile connections
or sterile docks are schematically shown and identified by reference numerals 31.
[0022] As further shown in Figure 1, system 10 may include sources of CO
2 and O
2 gas for delivery to chamber 19 of vessel 12 through gas-permeable membrane 18. Gas
sources 38 and 40 are fluidly connected to base docking unit 20 by gas lines 42 through
ports 44 in unit 20. The flow of gas may be controlled manually by gas control 45
or otherwise preprogrammed into controller 56 for automatic control.
[0023] System 10 may further include one or more sensors for monitoring the environment
within culturing vessel 12. As shown schematically in Figure 1, the interface area
46 between gas permeable membrane 18 and base docking unit 20 may house one or more
sensors as shown in Figure 1. Preferably, sensors are placed as close to chamber 19
to most accurately detect conditions within chamber 19. For example, system 10 may
be equipped with a pressure sensor 48 which measures the pressure at the gas permeable
membrane 18 and/or the composition of the gas delivered to vessel 12. A temperature
sensor 52 may also be included for determining or monitoring the temperature within
chamber 19 of vessel 12. Temperature sensor 52 may be configured to measure the air
temperature in the space between docking unit 20 and vessel 12. Alternatively, temperature
sensor 52 may provide for non-contact measurement (such as by non-contact infrared
sensors) to determine the temperature of vessel 12. If necessary, vessel 12 may further
be equipped with heating element 51 (Fig. 3) which, in response to an inadequate temperature
reading detected by sensor 52 may be activated and deliver additional heat to heat
the gases or the inner surface of vessel 12.
[0024] System 10 may further include a sensor for sensing the number of cells in chamber
19 of vessel and cell growth generally. Such a "cell enumeration" sensor 53 may be
a sensor whereby data is obtained in a non-invasive manner so that disturbance of
the cells or cell culture may be avoided. For example, in one embodiment, system 10
may include an optical detector that measures light reflectance and light scattering
properties of the cells and culture medium within chamber 19 to determine cell load.
As shown in Fig. 1, cell enumeration sensor 53 may be a reflection-based sensor and
emitter located in base docking unit 20. In this example, base 14 of vessel 12 may
be provided with a lens or other element aligned with sensor 53 to allow for transmission
of light through the bottom of vessel 20.
[0025] In another embodiment, base docking unit 20 may house (near the side of vessel 12)
a light emitting diode 55a of a known wavelength as well as linear or two-dimensional
sensor 55b or a CCD, as generally shown in Fig. 2. Whereas cell enumeration sensor
53, described above, may more directly determine the number of cells (e.g., cell density)
in vessel 12, the "side" array of sensors 55a (alone or in combination with 55b) generally
shown in Fig. 2 may be used to determine culture media components (such as glucose)
or cell culture by-products (such as lactose) which may be correlated to a cell number.
In a further alternative embodiment, sensor 55a may also be a reflection-based, combined
emitter and receiver as described above in connection with cell enumeration sensor
53. Other non-invasive means may include ultrasonic scanning of the cell culture or
measuring the capacitive properties of the cell culture.
[0026] In an alternative means for determining cell growth, vessel 12 may be equipped with
a sensor for measuring pH or dissolved oxygen in the culture. In another embodiment
of cell enumeration sensing, the system may be programmed to remove a sample of the
culture media through one of tubes 30a-30c by activation of pump 26 (syringe). The
sample may be analyzed by system 10 or analyzed offline to determine the state of
cell growth through either direct cell count or indirect means (glucose/lactate concentration).
The result may be entered into or electronically transferred to the system (controller
56) wherein the system may automatically (or by the operator) take appropriate action.
In any event, whether non-invasive or invasive means are used, the sensed cell enumeration
may provide feedback to controller 56 which may trigger reagent addition, agitation,
gas delivery, temperature adjustment or other system-controlled elements.
[0027] The functions of system 10 are in large part controlled by system controller 56 in
conjunction with a user interface 80 as shown in Figure 3. Controller 56 and user
interface 80 may be housed in console 76 or base docking unit 20. While controller
56 may take the form of one or more electrical components or circuits, controller
56 comprises a processor and an associated memory according to one embodiment. According
to such an embodiment, the processor may be programmed to carry out any of the actions
that controller 56 is described as being configured to perform below. The instructions
by which the processor is programmed may be stored on the memory associated with the
processor, which memory may include one or more tangible computer readable memories,
having computer executable instructions stored thereon, which when executed by the
processor, may cause the one or more processors to carry out one or more actions.
[0028] As further shown In Fig. 3, controller may be coupled to one more sensors 46, 52
and/or 55 as described above. Information or data regarding temperature, pressure
and cell enumeration is conveyed from the sensors to the controller 56. In response
to the data provided by the sensors, controller may, as necessary, adjust the rate
of delivery of CO
2 and/or O
2 gas to chamber 19 of vessel 12 from sources 38 and/or 40. As noted above, if the
temperature detected by heat sensor 52 is inadequate, controller 56 may be pre-programmed
to activate heating element 53. Controller 56 may also be programmed to activate the
motor of agitation assembly 22 or activate pumps 26 to deliver additional reagent
(medium) to vessel 12 in response to detected "cell enumeration" readings.
[0029] Thus, in accordance with the present disclosure, system may be pre-programmed to
deliver cells, media and supplements to vessel 12; control the conditions of the cell
culture including pH, temperature, % CO
2 and monitor cell growth (enumeration) without significant operator invention. Alternatively,
system 10 may be used in a more temporary fashion in conjunction with more traditional
cell culturing systems. In this example, vessel 12 could be removed from a conventional
incubator at a pre-set time, docked to base docking unit 20 so that above-described
sensors may be activated to take measurements, add or exchange media using the fluidic
controls of system 10 based on the sensed readings/measurements. Vessel 12 may then
be "undocked" from system 10 and returned to the incubator for further cell culturing
and ultimate harvesting.
OTHER EXAMPLES
[0030] Aspects of the present subject matter described above may be beneficial alone or
in combination with one or more other Aspects, as described below.
Aspect 1. A system for the culturing of biological cells including: a culturing vessel
having a top cap and a bottom support member defining a culturing chamber, the bottom
support member carrying a gas permeable membrane. The system further includes a base
assembly configured to receive the bottom support member in a gas-tight, mating manner,
the base assembly further including one or more channels for delivering to and/or
removing from the base one or more gases, and/or a culture medium source. The system
includes a control system with one or more sensors for sensing at least one of temperature
or pressure during cell culturing.
Aspect 2. The system of Aspect 1 further including one or more tubes joined to the
cap and extending into the culturing chamber, the one or more tubes defining a flowpath
for the culture medium.
Aspect 3. The system of any one of Aspects 1 or 2 wherein the base assembly further
includes an agitator.
Aspect 4. The system of Aspect 3 wherein the agitator includes a platform for tilting
the vessel.
Aspect 5. The system of any one Aspects 1 through 4 wherein the control system includes
a pressure sensor for monitoring gas pressure at the interface of the gas permeable
membrane and the base assembly.
Aspect 6. The system of any one of Aspects 1 through 5 wherein the base assembly further
includes a heating element.
Aspect 7. The system of any one of Aspects 1 through 6 wherein the system further
comprises a temperature sensor for measuring the temperature in the system.
Aspect 8. The system of Aspect 7 wherein the temperature sensor is located in the
base member.
Aspect 9. The system of Aspect 8 wherein the controller is configured to adjust the
temperature in the system in response to a reading from said temperature sensor.
Aspect 10. The system of any one of Aspects 1 through 9 further including a sensor
for measuring the content of one of or more gases in the system.
Aspect 11. The system of Aspect 10 wherein the sensor for measuring the content of
one of one or more gases is located in the base.
Aspect 12. The system of Aspect 11 wherein the controller is configured to adjust
the amount of one or more gases in response to a reading of the sensor for measuring
the content of the one or more gases.
Aspect 13. The system of any one of Aspects 1 through 12 further including sources
of CO2 and O2 gas.
Aspect 14. The system of Aspect 13 wherein the sources of CO2 and O2 gas are delivered to the base assembly by fluid lines joined to inlets in the base
assembly.
Aspect 15. The system of Aspect 14 wherein the controller is configured to control
the flow of gas to the base assembly.
Aspect 16. The system of any one of Aspects 1 through 15 further including one or
more pumping devices for delivering and withdrawing culture media and/or biological
cells to the culturing vessel.
Aspect 17. The system of Aspect 16 wherein the controller is configured to deliver
or withdraw one or both of the culture media and/or biological cells.
Aspect 18. The system of Aspect 3 wherein the controller is configured to actuate
the agitator during culturing.
Aspect 19. The system of any one of Aspects 1 through 18 including a cell enumeration
sensor.
Aspect 20. An automated method for culturing biological cells including monitoring
certain conditions within a culturing vessel having a gas-permeable membrane at the
base of said vessel. The method includes automatically adjusting the conditions based
on the monitoring.
Aspect 21. A method for automatically controlling the conditions of a biological cell
culturing environment including sensing the conditions of a biological cell culturing
with one or more sensors at or near the interface of a culturing vessel and base unit
for receiving said vessel; communicating selected conditions detected by said sensors
to a controller coupled to said base unit; and adjusting, as necessary, one or more
conditions of the culturing environment based on the detected selected conditions.
Aspect 22. The method of Aspect 21 including sensing the temperature at the interface
of the culturing vessel and the base unit.
Aspect 23. The method of Aspect 22 including adjusting the temperature at the interface.
Aspect 24. The method of Aspect 21 including sensing the temperature in the culturing
vessel.
Aspect 25. The method of Aspect 21 including measuring the number of cells and/or
cell growth in the vessel.
Aspect 26. The method of Aspect 25 including measuring the number of cells and/or
cell growth in the vessel by removing a sample of culture media from the vessel.
Aspect 27. The method of Aspect 21 including adjusting the delivery of gas to the
culturing vessel.
Aspect 28. The method of Aspect 21 further including replacing a culture medium in
the vessel.
Aspect 29. The method of Aspect 21 further including agitating the culturing vessel.
Aspect 30. The method of Aspect 29 including agitating the culturing vessel by tilting
the vessel.
[0031] The embodiments disclosed herein are for the purpose of providing a description of
the present subject matter, and it is understood that the subject matter may be embodied
in various other forms and combinations not shown in detail. Therefore, specific embodiments
and features disclosed herein are not to be interpreted as limiting the subject matter
of the invention.
1. A system for the culturing of biological cells comprising:
(a) a culturing vessel comprising a top cap and a bottom support member defining a
culturing chamber, said bottom support member carrying a gas permeable membrane;
(b) a base assembly configured to receive said bottom support member in a gas-tight,
mating manner, said base assembly further comprising one or more channels for delivering
to and/or removing from said base one or more gases;
(c) a culture medium source; and
(d) a control system comprising one or more sensors for sensing at least one of temperature
or pressure during cell culturing.
2. The system of Claim 1 further comprising one or more tubes joined to said cap and
extending into said culturing chamber, said one or more tubes defining a flow path
for said culture medium.
3. The system of any one of Claims 1 or 2 wherein said base assembly further comprises
an agitator.
4. The system of any one Claims 1 through 3 wherein said control system comprises a pressure
sensor for monitoring gas pressure at the interface of said gas permeable membrane
and said base assembly.
5. The system of any one of claims 1 through 4 wherein said base assembly further comprises
a heating element.
6. The system of any one of Claims 1 through 5 wherein said system further comprises
a temperature sensor for measuring the temperature in said system and said controller
is configured to adjust the temperature in said system in response to a reading from
said temperature sensor.
7. The system of any one of Claims 1 through 6 further comprising a sensor for
measuring the content of one of or more gases in said system and said controller is
configured to adjust the amount of said one or more gases in response to a reading
of said sensor for measuring the content of said one or more gases.
8. The system of any one of Claims 1 through 7 further comprising one or more pumping
devices for delivering and withdrawing culture media and/or biological cells to said
culturing vessel.
9. The system of Claim 8 wherein said controller is configured to deliver or withdraw
one or both of said culture media and/or biological cells.
10. The system of any one of Claims 1 through 9 comprising a cell enumeration sensor.
11. A method for automatically controlling the conditions of a biological cell culturing
environment comprising:
a) sensing the conditions of a biological cell culturing with one or more sensors
at or near the interface of a culturing vessel and base unit for receiving said vessel;
b) communicating selected conditions detected by said sensors to a controller coupled
to said base unit; and
c) adjusting, as necessary, one or more conditions of said culturing environment based
on said detected selected conditions.
12. The method of Claim 11 comprising sensing the temperature said culturing vessel.
13. The method of Claim 11 comprising measuring the number of cells and/or cell growth
in said vessel.
14. The method of Claim 11 comprising adjusting the delivery of gas to said culturing
vessel.
15. The method of Claim 11 further comprising replacing a culture medium in said vessel.